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An Improved Micropropagation Protocol for Manga Bamboo - Pseudoxytenanthera Stocksii (Munro) T.Q

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An Improved Micropropagation Protocol for Manga Bamboo - Pseudoxytenanthera Stocksii (Munro) T.Q Available online at www.worldnewsnaturalsciences.com WNOFNS 25 (2019) 141-154 EISSN 2543-5426 An Improved Micropropagation Protocol for Manga Bamboo - Pseudoxytenanthera stocksii (Munro) T.Q. Nguyen Bharat Singh Rajput1,2, Minal D. Jani1, K. Sasikumar1, M. Manokari3, M. S. Shekhawat4,* 1Tissue Culture Lab, Silviculture and Forest Utilization, Research Range, Rajpipla, Dist. Narmada, Gujarat, India 2Prem Sarita AgriBiotech, Rajpipla, Dist. Narmada, Gujarat, India 3Siddha Medicinal Plants Garden, Central Council for Research in Siddha, Mettur, TN, India 4Biotechnology Laboratory, K. M. Centre for P. G. Studies, Puducherry - 605 008, India *E-mail address: [email protected] ABSTRACT The present report illustrates an improved and reproducible micropropagation system for economically valuable bamboo species Pseudoxytenanthera stocksii using nodal segments. Direct organogenesis from nodal segments was accomplished by culturing on Murashige and Skoog (MS) medium supplemented with 6.0 mg/L 6-benzylaminopurine (BAP). On this medium combination 9.0±0.25 shoots per explant were induced. Shoot multiplication was subjective to the combination of auxins and cytokinins concentrations used and number of repeated transfer of mother explants or subculturing of in vitro regenerated shoot clumps on fresh medium. Maximum 41.9±1.00 shoots were observed on MS medium supplemented with 4.0 mg/L of BAP and 0.25 mg/L of naphthalene acetic acid (NAA). Half strength MS medium containing various concentrations of auxins was used for in vitro root formation. Successful rooting with maximum response was achieved on half strength MS medium + 50% sucrose and 1.0 mg/L IBA (24.3±0.27 roots). The rooted plantlets were planted the soilrite and hardened in the greenhouse for 6-8 weeks. Completely acclimatized plantlets exhibited 96% survivability. The present propagation protocol could satisfy the demand of P. stocksii and could be explored for the commercial propagation of this valuable manga bamboo. Keywords: In vitro propagation, Plant growth regulators, Dendrocalamus stocksii, Acclimatization ( Received 27 June 2019; Accepted 12 July; Date of Publication 13 July 2019) World News of Natural Sciences 25 (2019) 141-154 1. INTRODUCTION Bamboos belong to the grass family Poaceae, subfamily Bambusoideae and tribe Bambuseae. Bamboos stand as a valuable crop for the welfare of human as raw material for range of products and various industrial applications from time immemorial. Ecologically, bamboos support various forms of life and are preferred as important species in soil and water conservation, carbon sequestration, balancing O2 and CO2 in the atmosphere, barrier of ultra violet rays by reducing light intensity (Venkatachalam et al., 2015). Cultivation/ production of bamboo are foremost in India among the Asian countries. Across the globe, around 75 genera with approximately 1500 species were reported (FAO, 2001; Qing et al., 2008). In India, about 18 genera with 128 species have been reported and among them 20 species are under commercial cultivation (Seethalakshmi and Kumar, 1998). The bamboo dependent economy of India was estimated to be Rs. 26000 Crore by 2015 (Mahapatra and Shrivastava, 2013). The demand for bamboo is increasing than its production and Govt. of India has emphasized on the cultivation of bamboo in large scale on priority to support the developing industries with sufficient raw material. Conventionally, bamboo species are cultivated through macroproliferation through rhizomes, branch cutting or rooting of culms, which could not meet the huge demand (Adarsh Kumar, 1991; Banik, 1994). Commercially exploited woody bamboos are reported to be gregarious flowering group, hence the species/plant led to death followed by flowering, which leads to the extinction of various commercially important bamboo species. Lack of seed and/or other types of propagules could be a limiting factor for establishment of large-scale commercial plantations of the desired species. Hence, attention is needed to overcome the conventional propagation problems and tissue culture protocols could be necessary measures to satisfy the demand of propagules preparation in a short period of time (Banik, 2015). Several micropropagation protocols were developed in number of bamboo species all over the world viz., Gigantochloa atroviolaceae (Bisht et al., 2010), Dendrocalamus giganteus (Devi et al., 2012), Bambusa tulda, Melcocanna baccifera (Waikhom and Louis, 2014), Drepanostachyum falcatum (Saini et al., 2016), Bambusa bambos (Raju and Roy, 2016), Bambusa balcooa (Ansari et al., 2017), Dendrocalamus strictus (Rajput et al., 2019) etc. One such significant domesticated bamboo species explored enormously and being in demand is Pseudoxytenanthera stocksii, commonly known as Manga Bamboo, endemic to central Western Ghats (Singhal and Gangopadhyay, 1999). It is an economically important and multipurpose bamboo, recommended by National Bamboo Mission (NBM) amongst the top 20 bamboo species of India. It grows up to 25 m, which is used extensively as building material for making house construction, scaffolding, basketry, furniture making, crafts, stakes and agri tools (Nath and Das, 2008; Viswanath et al., 2013). As per the report by Rane et al. (2016), In India, the potential revenue estimated about Rs. 4.5 lakhs (USD 6705)/year/ha from culms. Farmers earn Rs. 50/- to 60/- from each stick and in general, 10 to 15 sticks used to be harvested from each clump, so there is massive need of planting material. P. stocksii are characterized by the presence of glabrous, solid, grey-green stem with a pubescent ring. Its leaves varies in size (10-20 x 1.2-2 cm), petiolate with setaceous tip, rounded base, narrow midrib and toothed ligule. Spikelets range 1 cm long, glabrous, flower glumes 2 ovate, sub-acute and dorsally mucronate. Ovary is ovoid with long and slender style, grains elongate, beaked and smooths (Rane, 2015). The nutritional analysis of P. stocksii reveals the presence of protein, fat, carbohydrate, fibre and the cyanogenic glucosides, which causes -142- World News of Natural Sciences 25 (2019) 141-154 bitterness is comparatively less in P. stocksii (AOAC, 1998, 2005; Rane et al., 2016). Its leaf was found to have allelopathic effect on Arachis hypogaea (Rawat et al., 2018). The various synonyms of Pseudoxytenanthera stocksii are Dendrocalamus stocksii (Munro) M.Kumar, Remesh & Unnikrishnan, Gigantochloa stocksii (Munro) T.Q. Nguyen, Oxytenanthera stocksii Munro, Pseudotenanthera stocksii (Munro) R.B. Majumdar and Pseudoxytenanthera stocksii (Munro) H.B. Naithani (The Plant List, 2019). P. stocksii is commonly propagated by seeds and culm cuttings, but the major hindrance to the species is the production of sterile flowers and absence of viable seeds. Due to the growing concern in this species, the prime objective of the study was to evaluate various factors affecting shoot proliferation and successful establishment of P. stocksii to be adapted for commercial propagation. 2. MATERIALS AND METHODS 2. 1. Selection of plant material and disinfection Freshly emerged nodal shoot segments from 6 years old physically healthy plants of P. stocksii were collected from the Vadia Palace Forest Campus, Rajpipla during June 2018 to February 2019. The nodal segments measuring 2.0-3.0 cm in length were used as explants. The explants were treated with 0.1% (w/v) solution of systemic fungicide (Bavistin) for 5-10 min and washed with autoclaved distilled water. The explants were disinfected using 0.1% (w/v) mercuric chloride (HgCl2) for 5-10 min and thereafter washed with autoclaved distilled water for 6-10 times to remove the traces of HgCl2 from the explants. 2. 2. Culture medium and in vitro culture conditions Cultures were established with MS basal medium (Murashige and Skoog, 1962) supplemented with 0.8% agar and additives (50 mg/L ascorbic acid, 25 mg/L each of adenine sulphate, L-arginine and citric acid). The pH of nutrient medium was attuned to 5.8 ±0.02 using 0.1 N NaOH or HCl prior to steam sterilization at 1.06 kg cm-2 pressure for 15 min at 121 ºC temperature. All the cultures were incubated at 16/8 h/d light/dark period in a growth room maintained with 40–50 µmol m-2s-1 Spectral Flux Photon Density (SFPD) light intensity, provided by cool and white fluorescent tubes (Philips, India) at 28 ±2 ºC temperature and 60- 70% relative humidity (RH). 2. 3. Culture induction and multiplication of shoots The surface sterilized explants dissected to an appropriate size (2-3.5×0.5-1.5 cm) were inoculated horizontally and vertically on MS medium enriched with additives (50 mg L-1 ascorbic acid and 25 mg L-1 each of adenine sulfate, citric acid and arginine), 3% (w/v) sucrose, gelled with 0.8% (w/v) agar and supplemented with various concentrations (2.0-10.0 mg L-1) of cytokinins (6-benzylaminopurine [BAP] and 6-furfurylaminopurine [Kn]). The shoots emerged from the nodes were periodically subcultured after every two-four weeks time interval on MS medium augmented with same concentration of cytokinins. The MS medium devoid of PGRs treated as control. Shoots were multiplied by repeated transfer of mother explants with freshly regenerated shoots on the fresh MS medium. These were also multiplied by the transfer of in vitro raised shoot clumps on the fresh medium and cultured on MS medium augmented -143- World News of Natural Sciences 25 (2019) 141-154 with different concentrations and combinations of cytokinins and auxins for 4 weeks in culture vessels and incubated at 25±2 ºC temperature under 12 h/d photo period with 50 µmol/m2/s PPFD light intensity. 2. 4. In vitro rooting of the shoots and Acclimatization of plantlets The actively growing shoots from the multiplication stage were separated from the culture cluster and inoculated on different strengths of MS medium (full, half and one-fourth) incorporated with various concentrations of auxins (Indole-3-acetic acid [IAA], indole-3- butyric acid [IBA] and 1-Naphthaleneacetic acid [NAA]) (0.5-3.0 mg/L) for rooting under in vitro conditions. The rooted shoots were transferred to nursery pro trays (diameter 10 cm) containing sterile soilrite®, moistened with one-fourth aqueous MS salts and maintained in the greenhouse for 4 week.
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